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Multisite phosphorylated peptide (protein) recognizing compound and detection method, imaging method, alzheimer's disease diagnosing method and reagent kit using the same

Active Publication Date: 2009-09-17
CANON KK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]The compound for detecting a phosphorylated protein or peptide of the present invention can detect a captured phosphorylated substance specifically and rapidly with high sensitivity because the compound of the present invention can recognize phosphorylation sites of the protein or peptide specifically to the distance between the phosphorylation sites and bind to the peptide. As a result of the binding of the compound and the protein or peptide, a chromogenic or luminescent change is induced in the compound, and the phosphorylated protein or peptide can be detected or a kinase activity can be measured with high sensitivity by measuring this change. Since the compound of the present invention has a property of selectively recognizing a protein or peptide phosphorylated at multiple specific sites and binding to the phosphorylated protein or peptide, the compound of the present invention can also be used as means for isolating or purifying a multisite phosphorylated protein or peptide. The compound for detecting a phosphorylated protein or peptide of the present invention also has a potential as a molecular tool for elucidating intracellular signal transduction mechanisms or as an inhibitor of an interaction between proteins via a specific phosphate group. The present invention provides a novel compound for detecting an excessively phosphorylated tau protein utilizing ability to specifically recognize a phosphorylated protein or peptide and a method for diagnosing Alzheimer's disease using the same.

Problems solved by technology

In this technique, phosphorylated proteins can be detected with high sensitivity, but facilities for using radioactive isotopes are required, and there are problems of cumbersome operations, exposure, and contamination.
This technique suffers from problems that an antibody recognizing a target protein must be obtained as a precondition, and cumbersome operations are required.
This detection method suffers from low precision.
Antibodies are excellent compounds in view of specificity, but costs for producing antibodies are problematic.
Furthermore, in intracerebral imaging, poor delivery of antibodies into the brain is a serious problem since their molecular weights are as large as 150 kDa.
However, since these compounds do not greatly change their affinity due to the difference in the distance between phosphate groups, it is difficult to recognize specific multisite phosphorylation sites using the distance between the phosphate groups as a reference.

Method used

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  • Multisite phosphorylated peptide (protein) recognizing compound and detection method, imaging method, alzheimer's disease diagnosing method and reagent kit using the same
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  • Multisite phosphorylated peptide (protein) recognizing compound and detection method, imaging method, alzheimer's disease diagnosing method and reagent kit using the same

Examples

Experimental program
Comparison scheme
Effect test

example 1

Synthesis of Luminescent Compound

[0080]As the luminescent compound of the present invention, a Zn(Dpa)-stilbazole complex 1 (FIG. 3) represented by the above-mentioned formula (16) was synthesized by the following scheme. For comparison, a compound 2 having one Dpa (FIG. 4) was also synthesized. FIG. 5 illustrates a synthesis scheme.

Example 1(1)

Synthesis of Compound 3-1

[0081]5.0 g (31.7 mmol) of 2-pyridinecarboxylic acid hydrochloride and 15 mL of thionyl chloride were placed in a 100-mL three-neck recovery flask and stirred on an ice bath. 0.5 mL (31.7 mmol / l eq) of distilled water was slowly added dropwise to this mixture solution, and then reflux with heating was started. The reaction was followed by TLC (hexane / EtOAc=1 / 1), and completion of the reaction was confirmed three days later. The solvent was evaporated under reduced pressure, toluene was further added, and the mixture was evaporated under reduced pressure. When 30 mL of toluene was added to the residue, the mixture was ...

example 1 (

Example 1(17)

Conclusion

[0100]In summary, to make the skeleton of a compound molecule itself rigid, a Zn / Dpa complex was allowed to bind directly to a spacer (vinyl group), and a novel stilbazole compound in which the spacer was directly introduced at the fourth position of a pyridine ring in the Dpa was synthesized (compounds 1 and 2). Compound 1 recognizes and is crosslinked to phosphate groups existing distantly at the positions of (i, i+2) on the multisite phosphorylation sequence of a tau protein and binds in ratio of 1:1, and phosphorylation was successfully identified with dual wavelength fluorescence changes (Ka=˜105 M−1). Furthermore, it was found that compound 1 did not bind to two phosphate groups existing at the positions of (i, i+4) or (i, i+6) among a plurality of phosphate groups that existed and selectively recognized only two phosphate groups at the positions of (i, i+2). In a multisite phosphorylated peptide-recognizing compound in which a spacer binds to a nitrogen...

example 2

Synthesis of Luminescent Compound (BODIPY-Zn(Dpa))

[0102]As another luminescent compound according to the present invention, BODIPY-Zn(Dpa) represented by the formula (12) described above (compound 9) was synthesized as follows. FIG. 14 illustrates a synthesis scheme.

Example 2(1)

Synthesis of Compound 11

[0103]1.0 g (7.24 mmol) of 3,5-dihydroxy-benzaldehyde, 2.5 g (18.1 mmol, 2.5 eq) of potassium carbonate, 628.7 mg (7.24 mmol, 1.0 eq) of lithium bromide and 35 mL of dry DMF were placed in a 100-mL three-neck recovery flask, and the mixture was stirred at 100° C. Then, a solution of triethylene glycol monochlorohydrin in dry DMF (2.31 mL (15.9 mmol, 2.2 eq) / 10 mL) was added dropwise to the reaction solution, and the mixture was stirred continuously at 100° C. The reaction was followed by TLC (CHCl3 / MeOH=10 / 1), and completion of the reaction was confirmed five days later. Insoluble matters were isolated by filtration, and the filtrate was evaporated under reduced pressure. Ethyl acetate...

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Abstract

There are provided a novel compound which captures a multisite phosphorylated peptide or protein specifically to a phosphorylation site and a method for detecting a multisite phosphorylated peptide or protein using the same. In particular, there are provided a compound which specifically detects an excessively phosphorylated tau protein observed in the brain affected by Alzheimer's disease and a method for diagnosing Alzheimer's disease in vitro or in vivo using the compound. By bringing a metal complex compound having two dipicolylamine (Dpa) moieties and a spacer including a chromogenic or luminescent functional group or atom group into contact with a multisite phosphorylated peptide or protein, the compound recognizes the distance between phosphate groups and specifically binds to the peptide or the protein, and a multisite phosphorylated peptide or protein or a kinase activity is optically detected by measuring the change, or a multisite phosphorylated peptide or protein or kinase activity is imaged by an optical imaging method applying the change in the luminescence.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention relates to a compound for detecting a phosphorylated peptide or protein that recognizes a phosphate group at multiple sites. Furthermore, the present invention relates to a method for detecting a multisite phosphorylated peptide or protein in a sample using the compound. In particular, the present invention relates to a compound that specifically recognizes an excessively phosphorylated tau protein, a method for detecting a phosphorylated tau protein in a sample using the compound, and a method for detecting Alzheimer's disease, an imaging method, a method for diagnosing Alzheimer's disease and a reagent kit using the compound.[0003]2. Description of the Related Art[0004]Proteins in the body are subjected to various biochemical modifications and change their higher-order structures, functions and activities to regulate biological function. Protein phosphorylation, one of protein modifications, is a...

Claims

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Application Information

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IPC IPC(8): G01N33/68C07D213/02C07F3/06C07F5/02
CPCC07D207/335C07D401/14Y10T436/163333G01N33/6842Y10T436/16C07F5/022A61P25/28
Inventor HAMACHI, ITARUYAMAUCHI, FUMIOYANO, TETSUYAYOSHIMURA, KIMIHIROOJIDA, AKIOSAKAMOTO, TAKASHIINOUE, MASAAKI
Owner CANON KK
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